Agilent 1260 series hplc system

200 μL of urine sample was transferred into a 15-mL polypropylene (PP) tube and spiked with the labeled internal standard mixture (2-¹³C, 99%; ¹⁵N, 98+% Glyphosate), allowed to stand at room temperature for 30 min and then diluted to 1.0 mL (5-fold dilution) with 1% formic acid in water. The diluted sample was vortexed for 1 min, centrifuged and filtered through a nonsterile regenerated cellulose (RC) membrane filters (0.2 μm; Phenomenex, Inc., Torrance, CA, USA). The filtrate then transferred into an auto sampler vial for LC-MS/MS analysis. An Agilent 1260 Series HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) coupled with an ABSCIEX 4500 QTRAP mass spectrometer (Applied Biosystems, Foster City, CA, USA) running under negative mode electrospray ionization was used for the analysis. An anion-exchange column, Dionex IonPac AS 21 (2 mm × 250 mm, 7 μm) was employed for the separation of target chemicals under isocratic elution condition with a mobile phase consist of 1% formic acid in water and acetonitrile (95:5) mixture. Isotopic dilution mass spectrometry with MRM mode of analysis was used for selective quantification of the target chemicals. The QA/QC protocols included matrix spike (mean spike recoveries (n=5) for glyphosate was 109.5%) and procedural blanks (non-detected or

The reagents and solvents were purchased from commercial sources (Fisher) and used directly. The intermediates and products were purified by using an ISCO CombiFlash system and prepacked SiO₂ cartridges. Final compounds were purified on preparative high-pressure liquid chromatography (RP-HPLC) was performed on Agilent 1260 Series system. Systems were run with 0–20% methanol/water gradient with 0.1% TFA. NMR spectra were acquired on a Bruker AV500 instrument (500 MHz for ¹H-NMR, 126 MHz for ¹³C-NMR). TLC-MS were acquired using Advion CMS-L MS. Matrix-assisted laser desorption ionization mass spectra (MALDI-MS) data were acquired in positive-ion mode using a Sciex 4800 MALDI TOF/TOF MS. The peptides were synthesized using a CEM Liberty Blue Automated Microwave Peptide Synthesizer with the manufacturers standard coupling cycles at 0.1 mmol scale. The purity of final compounds was confirmed by Waters LC-MS system and/or Agilent 1260 Series HPLC system. Systems were run with 0–40% methanol/water gradient with 0.1% TFA. All the purity of target compounds showed >95% in RP-HPLC.

The HPLC system consisted of an Agilent 1260 Series HPLC system (Agilent Technologies, Palo Alto, CA, USA), a Shimadzu, System Controller, SCL-10A Vp, Pump, LC 10AD Vp Solvent Degasser (DGU14A, Shimadzu, Columbia, MD, USA), and an CTC PAL autosampler (Zwingen, Switzerland). The analytical column was XBridge C₁₈, 50 × 2.1 mm, 5µm (Waters Corp, Milford, MA, USA). The column temperature was maintained at 25 °C. Mobile phase A was water and mobile phase B was 0.1 % formic acid in acetonitrile. The elution pump (Agilent 1260) gradient was: 0–1.0 min 0.0% B at 0.4 mL min⁻¹ flow, 1.1–3.0 min from 50% to 90% B at 0.4 mL min⁻¹ flow rate, 3.1–7.0 min 90% B at 0.8 mL min⁻¹ flow rate and 7.1–10.0 min 0.0% B at 0.8 mL min⁻¹ flow rate. The makeup pump (Shimadzu) ran an isocratic gradient at 50:50 mobile phase A: mobile phase B and at flow rate of 0.2 mL min^-1. The switching valve program was: 0–1.8 min at position A (loading sample into column and desalting), 1.8–3.0 min at position B (elution to MS source) and 3.0 to 10.0 min at position A (column flushing and re-equalibration). Injection volume was 50 µL and total run time was 10.0 min.